Immunomodulatory compounds

ABSTRACT

Compounds are described which modulate the tzrosine kinase activity of p56 lck  and signal transduction pathways in which this enzyme is involved. The invention also relates to compounds which have immunomodulatory activity, e.g., which have immunosuppressant or immunostimulatory activity, and/or which have an antineoplastic effect. The invention further relates to compositions comprising these compounds, and methods of using them. Compounds are described which modulate the tyrosine kinase activity of p56.

This application is filed under 35 U.S.C. 371 based on internationalapplication PCT/US01/41467, filed Jul. 31, 2001, and claims benefit ofU.S. Provisional Application Ser. No. 60/221,687, filed Jul. 31, 2000,the disclosures of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

This invention relates, e.g., to compounds which modulate the tyrosinekinase activity of p56^(lck) and signal transduction pathways in whichthis enzyme is involved. The invention also relates to compounds whichhave immunomodulatory activity, e.g., which have immunosuppressant orimmunostimulatory activity, and/or which have an antineoplastic effect.The invention further relates to compositions comprising thesecompounds, and methods of using them.

DESCRIPTION OF THE INVENTION

In one embodiment, the invention relates to a method of achieving animmunomodulatory effect in a patient in need thereof, comprisingadministering an effective amount of a compound of the formula

wherein

X is sulfur, SO₂, SO, methylene, oxygen, carbonyl, ethylene, amide,ester or thioester,

R₁ is a negative charge functional group,

R₂ is an electronegative group, which can be an amine, amide, urea,carbamide, carbonate, anhydride, thioamide, thiourea, thiocarbamide,thiocarbonate, thioanhydride, hydroxyl, or an ester, e.g., an alkylester, acyl ester, aryl ester, alkyl thioester, acyl thioester or arylthioester,

Y₁, Y₂, Y₃, Y₄, Y₅, Y₆, Y₇, Y₈, Y₉, and Y₁₀ are, independently,hydrogen, methyl, ethyl, propyl, isopropyl or halogen, and

optionally, one or more of the phenyl aromatic rings are replaced by afused aromatic ring, heteroaromatic ring, or fused heteromatic ring, forexample, by replacing 1-3 carbon atoms of one of more of the aromaticrings with N atoms. (Examples include naphthyl, pyridinyl, quinolinyland isoquinolinyl rings),

or of the formula

wherein

X and Y₁ to Y₇ are as defined above,

R₁ and R₄ are the same or different, and are as defined above for R₁,

R₃ is hydrogen, halogen hydroxyl, amine, methyl, ethyl, propyl orisopropyl, and

optionally, one or more of the phenyl aromatic rings are replaced by afused aromatic ring, heteroaromatic ring, or fused heteromatic ring, forexample, by replacing 1-3 carbon atoms of one of more of the aromaticrings with N atoms. (Examples include naphthyl, pyridinyl, quinolinyland isoquinolinyl rings),

or of the formula

wherein

X and R₁ are as defined above, and Y₁ to Y₁₂ are as defined above for Y₁to Y₁₀,

X₁ is a sulfur, SO₂, SO, methylene, oxygen, carbonyl, ester, thioesteror amide,

R₃ and R₅ are the same or different, and are as defined above for R₃,and

optionally, one or more of the phenyl aromatic rings are replaced by afused aromatic ring, heteroaromatic ring, or fused heteromatic ring, forexample, by replacing 1-3 carbon atoms of one of more of the aromaticrings with N atoms. (Examples include naphthyl, pyridinyl, quinolinyland isoquinolinyl rings),

or a pharmaceutically acceptable salt thereof.

Preferably, the negative charge functional group of the compounds aboveis a —COOH, amide, ester, nitro, phosphate or sulfate radical (byphosphate is meant esters of phosphorous based acids, such asphosphorous or phosphoric acid; by sulfate is meant esters ofsulfur-based acids, such as sulfonic or sulfinic acid); the hydrogenbond acceptor is preferably a carboxylic acid or an ester of carboxylicacid, e.g., an alkyl ester, acyl ester, aryl ester, alkyl thioester,acyl thioester or aryl thioester.

In a most preferred embodiment, the compound is

In exemplary embodiments, X₁ and X are, independently, O, S, thioesteror carbonyl, R₁ (and independently R₄) is COOH or nitro,

R₂ is an alkyl ester, amino or OH,

R₃ (and independently R₅) is H, Cl, F, OH or methyl,

the Y's are independently H, methyl, F or Cl, and the “phenyl” ringsremain phenyl rings or are naphthyl or pyridinyl.

In another embodiment, the invention relates to a method for achievingan antineoplastic effect in a patient in need thereof, comprisingadministering an effective amount of a compound of formulas I, II or IIIas noted above.

In another embodiment, the invention relates to a method of modulatingthe binding of a p56^(lck) molecule via an SH2 domain thereof to acorresponding cellular binding protein, and/or modulating the activityof a p56^(lck) molecule via binding to an SH2 domain thereof, comprisingbinding to an SH2 domain of said p56^(lck) molecule to a compound offormulas I, II or III, and more preferably, compounds of formulas I′,II′ or III′, as noted above.

In another embodiment, the invention relates to a method for achievingan antineoplastic effect in a patient in need thereof, comprisingadministering an effective amount of a compound of formulas I′, II′, orIII′.

In another embodiment, the invention relates to a method of inhibitinghyperproliferative cell growth in a patient in need thereof, comprisingadministering an effective amount of a compound of formulas I, II, III,I′, II′, or III′.

In another embodiment, the invention relates to a method of achieving animmunomodulatory effect in a patient in need thereof, comprisingadministering an effective amount of a compound of formulas I′, II′, orIII′.

The compound of formula I′ above (compound I′) has a molecular weight ofabout 352 and the chemical formula, C₂₀H₁₆O₄S. It activates p56^(lck)kinase activity and results in stimulation and activation of T-cells.Compound I′ can be used, e.g., as an immunostimulant.

The compound of formula II′ above (compound II′) has a molecular weightof about 305 and the chemical formula, C₁₄H₈O₅ClN. It activatesp56^(lck) kinase activity at low concentrations (doses) and inhibitsactivity at high concentrations (doses). Compound II′ can be used, e.g.,as an immunostimulant or as an immunosuppressant, or as anantineoplastic agent, depending on the concentration (dosage) of thecompound.

The compound of formula III′ above (compound III′) has a molecularweight of about 421 and the chemical formula, C₁₈H₁₀C₁₂N₂O₄S. Itinhibits p56^(lck) kinase activity and can be used, e.g., as animmunosuppressant or as an antineoplastic agent.

In a preferred embodiment, a compound of the invention does not comprisea phosphotyrosine or related moiety.

All compounds can be prepared fully conventionally, using known reactionchemistry, starting from known materials or materials conventionallypreparable. [See, e.g., Houben-Weyl, Methoden der Organischen Chemie[Methods of Organic Chemistry], Georg-Thieme-Verlag, Stuttgart]. Forexample, a compound of the invention can be synthesized via a SN_(AR)reaction of the species,

Many compounds of the invention are readily available from standardsources, such as chemical supply houses, or can be generated fromcommercially available compounds by routine modifications such as thosedescribed above. For example, compounds I, II and III noted above can beobtained from the Maybridge Chemical Company, LTD (Maybriege PLC;Trevillet, Tintagel; Cornwall, PL34 OHW; England). Compound I′ isMaybridge #BTB11478; compound II′ is Maybridge #SB00529; and compoundIII′ is Maybridge #SPB01890.

Among the advantages of the compounds of the invention are that themolecules are not susceptible to enzymatic hydrolysis (as are certainpeptide and protein modulators of protein tyrosine kinase activity), andthat they exhibit good cell permeability characteristics.

Without wishing to be bound to any particular mechanism, this inventionrelates, e.g., to compounds that interact specifically with proteins,e.g., protein tyrosine kinases, which are involved in intracellularsignaling pathways, in particular to compounds that interact with SH2domains of such tyrosine kinases, and more particularly to compoundsthat interact with an SH2 domain of the p56^(lck) src family tyrosinekinase. Among other functions, the p56^(lck) protein is involved insignal transduction pathways involved in T cell antigen receptoractivation signaling required for mounting an active immune response,and in aspects of cell proliferation, e.g., proliferation of neoplasticcells. It is proposed that compounds of the invention, by interactingwith p56^(lck), particularly with an SH2 domain thereof, modulate thekinase activity of the protein and/or modulate its ability to interactwith a corresponding cellular binding protein, and thereby modulateimmune responses, directly or indirectly, and neoplastic cellproliferation. Compounds of the invention can either enhance or inhibitsignal transduction pathways, including downstream signal transductionprocesses in a signal transduction pathway, or they can be biphasic,either enhancing or inhibiting, depending on conditions. The effect ofany given compound can be routinely determined by screening in one ormore of the assays described herein or other fully conventional assays.

The non-catalytic domains of p56^(lck) kinase, e.g. the SH2 domain(s),mediate specific intramolecular and intermolecular interactions that areimportant for the regulation of p56^(lck) function; they exert bothnegative and positive effects on kinase activity. In general, theintramolecular interaction keeps p56^(lck) in an inactive state, and theintermolecular interactions facilitate p56^(lck) kinase action. Forexample, the SH2 domain can positively regulate p56^(lck) enzymaticactivity by targeting p56^(lck) to specific cellular sites [ITAM(immunoreceptor trosine based activation motifs) phosphotyrosines] wheresubstrate phosphorylation is needed; and p56^(lck) that is bound tophosphtyrosine sites via its SH2 domain can exhibit higher enzymaticactivity, thereby enhancing further phosphorylation of substrates.Without wishing to be bound to any particular mechanism as to how thisis accomplished, it is proposed that the compounds which bind to the SH2domain can either increase (activate, enhance, stimulate), decrease(suppress, inhibit, depress), or have no effect on, kinase activity andattendant cellular phosphorylation events (e.g., processes involved inintracellular signaling).

p56^(lck) plays an important role in modulating immune responses.p56^(lck) is a T-cell specific kinase, the majority of which isassociated with CD4 (in T_(H) cells) and CD8 (in cytotoxic T cells). Thep56^(lck) kinase is responsible, e.g., for an early step in activating Tcells—the phosphorylation of ITAM in CD3 chains—which in turn initiatesmultiple intracellular cascades of biochemical events leading to, e.g.,actin polymerization, enhanced gene transcription, cellularproliferation and differentiation. p56^(lck) also plays an importantrole in a second important step in the activation of Tcells—immunological synapse formation. The compounds of the inventioncan modulate the immune response by, e.g. modulating T-cell activation,or indirectly by modulating downstream processes of a signaltransduction pathway. As used in this application, the term “modulate”means to change, e.g., to increase (activate, enhance, stimulate) ordecrease (suppress, inhibit, depress) a reaction or an activity.Compounds of the invention can be said to modulate the binding of ap56^(lck) SH2 domain to a “corresponding cellular binding protein,”which term, as used herein, refers to any cellular binding protein whosebinding to p56^(lck) is mediated by SH2 domains. Such correspondingcellular binding proteins include, e.g., CD3 chains, ZAP-70, p62, Lad,CD45, Sam68 or the like.

Many protein tyrosine kinases play a role in regulating cellular events,including gene activation and/or regulation, and thus, e.g., in cellproliferation. p56^(lck) is a proto-oncogene, which has been implicatedin a number of pathological conditions that involve undesirablehyperproliferation of cells. For example, overexpression ofconstitutively active p56^(lck) has been observed in murine and humanlymphomas, suggesting that p56^(lck)-mediated phosphorylation ofcellular proteins stimulates lymphocyte proliferation. In addition,overexpression and activation of p56^(lck) appears to play an importantrole in the human lymphoid cell transformation induced by Epstein-Barrvirus and Herpesvirus Saimiri. Moreover, transgenic mice overexpressingwild type p56^(lck) and a constitutively active form of p56^(lck) inthymocytes develop thymoma, suggesting that even the overexpression ofwild type p56^(lck) can transform cells under these conditions.Compounds of the invention, e.g. compounds which inhibit p56^(lck)activity, are useful for the treatment of conditions involvinghyperproliferative cell growth, either in vitro (e.g., transformedcells) or in vivo. Conditions which can be treated or prevented by thecompounds of the invention include, e.g., a variety of neoplasms,including benign or malignant tumors, a variety of hyperplasias, or thelike. Compounds of the invention can achieve the inhibition and/orreversion of undesired hyperproliferative cell growth involved in suchconditions.

As used herein, the term “hyperproliferative cell growth” refers toexcess cell proliferation. The excess cell proliferation is relative tothat occurring with the same type of cell in the general populationand/or the same type of cell obtained from a patient at an earlier time.“Hyperproliferative cell disorders” refer to disorders where an excesscell proliferation of one or more subsets of cells in a multicellularorganism occurs, resulting in harm (e.g., discomfort or decreased lifeexpectancy) to the multicellular organism. The excess cell proliferationcan be determined by reference to the general population and/or byreference to a particular patient (e.g., at an earlier point in thepatient's life). Hyperproliferative cell disorders can occur indifferent types of animals and in humans, and produce different physicalmanifestations depending upon the affected cells. Hyperproliferativecell disorders include, e.g., cancers, blood vessel proliferativedisorders, fibrotic disorders, and autoimmune disorders.

Activities and other properties of the compounds of the invention (andcomparisons of those activities to those of art-recognized, comparisoncompounds) can be measured by any of a variety of conventionalprocedures.

A variety of in vitro assays can be used to measure biological and/orchemical properties of the compounds, and are conventional in the art.For example, in vitro binding studies can determine the affinity and thespecificity of binding of the compounds, e.g., to a p56^(lck) SH2domain. Example 4 illustrates a method to determine K_(D) and IC₅₀values, using tritiated compounds and purified, recombinant p56^(lck)SH2 domains. Similar assays can show that compounds bind selectively invitro to a particular site, e.g., to the p56^(lck) SH2 domain, but notto other sites, e.g., Hck, Fyn, Src, Shc or ZAP-70 SH2 domains. Example5 illustrates an in vitro co-immunoprecipitation (IP) kinase assay.Again, similar assays can show the specificity of binding of thecompounds. Example 6 illustrates an assay to determine specificity ofthe binding.

Other conventional in vitro assays can measure the effect (e.g.,inhibition or enhancement) of the compounds on biological activitiesassociated with tyrosine protein kinases, e.g., p56^(lck). p56^(lck)activities which are involved in immune responses include, e.g., thephosphorylation of, e.g., tyrosine in the ITAM consensus sequencepresent in certain molecules, e.g., CD3 chains; immunological synapseformation, e.g., with corresponding cellular binding proteins; or thelike. Example 1 illustrates an in vitro assay for Jurkatcell-activation-dependent phosphorylation, an activity that iscorrelated with T-cell activation. Compound I′ is shown to stimulate thephosphorylation, and compounds II′ (at high dosage) and III′ to beinhibitory. The effects are shown to be dose-dependent. Example 2illustrates an in vitro assay for cell viability, which indicates if acompound is cytotoxic or cytostatic. Compound III′ is shown to exhibit areversible inhibition of Jurkat cell growth. Example 3 illustrates an invitro assay for IL-2 production, an activity which is correlated withT-cell activation. Compound III′ is shown to inhibit IL-2 production inOKT-3 treated Jurkat cells. Example 7 illustrates a mixed lymphocyteculture assay.

A variety of in vivo assays can be used to demonstrate immunomodulatoryproperties of the compounds. Such in vivo assays, and appropriate animalmodels for disease conditions that can be treated with the compounds,are well-known to those of skill in the art. For example, animal modelsfor rheumatoid arthritis are illustrated in Example 8.

Assays to measure the effect of compounds (e.g., phosphotyrosine kinaseinhibitors) on cell growth proliferation) and cell transformation areconventional. A variety of typical assays are described, e.g., inKelloff, G. J., et al., Cancer Epidemiol Biomarkers Prev., 1996. 5(8),p. 657-66; Wakeling, A. E., et al., Breast Cancer Res Treat, 1996,38(1), 67-73; Yano, S., et al., Clin Cancer Res, 2000, 6(3), p. 957-65;Reedy, K. B., et al., Cancer Res, 1992, 52(13), p. 3636-41; Peterson, G.and S. Bames, Prostate, 1993; 22(4), p. 335-45; Scholar, E. M. and M. L.Toews, Cancer Lett, 1994, 87(2); 159-62; Spinozzi, F., et al., Leuk Res,1994, 18(6), p. 431-9; Kondapaka, B. S. and K. B. Reddy, Mol CellEndoctinol, 1996, 117(1), p. 53-8; Moasser, M. M., et al., Cancer Res,1999, 59(24), p. 6145-52; Li, Y., M. Bhuivan & F. H. Sarkar, Int JOncol, 1999, 15(3), p. 525-33; Baguley, B. C., et al. Eur J Cancer,1998, 34(7), p. 1086-90; and Bhatia, R., H. A. Munthe, and C. M.Verfaillie, Leukemia, 1998, 12(11), p. 1708-17.

Variations of the assays described herein, as well as other conventionalassays, are well known in the art. Such assays can, of course, beadapted to a high throughput format, using conventional procedures.

Moreover, conventional methods of computer-aided rational drug designcan provide an indication as to whether an inventive compound has theproper “fit” to, and is complementary to, a region of the protein whichis important for specificity of binding, e.g., a p56^(lck) SH2 domain,as opposed to, e.g., Hck, Fyn, Src, Shc or ZAP-70 SH2 domains. Inparticular, such methods can indicate whether a compound iscomplementary to the pY+3 binding site of p56^(lck). The terms “specificbinding” or “specificity of binding” as used herein mean that aninventive compound interacts with, or forms or undergoes a physicalassociation with, a particular SH2 domain (e.g., a p56^(lck) SH2 domain)with a higher affinity, e.g., a higher degree of selectivity, than forother protein moieties (e.g., SH2 domains of other protein kinases).Furthermore, properties of the compounds, such as, e.g., solubility,chemical stability and the absence of chemical groups known to imparttoxicity, can be analyzed on the basis of known properties of certainchemical substituents, which are well known to those of skill in theart. See, e.g., Opera, J. Comput.-Aided Mol. Des., 2000. 14: p. 251-264.

The compounds of the invention are effective for binding to, e.g.,p56^(lck) SH2 domains, and for modulating the activity of, e.g.,p56^(lck) in animals, e.g., mammals, such as mouse, rat, rabbit, pets,(e.g., mammals, birds, reptiles, fish, amphibians), domestic (e.g.,farm) animals, and primates, especially humans. The inventive compoundsexhibit, e.g., immunomodulatory activity and/or antineoplastic activity,and are effective in treating diseases in which, e.g., aberrantregulation or activity of tyrosine kinase (e.g., p56^(lck)) and/orintracellular signaling responses are involved. For example, compoundswhich stimulate immune responses (immunostimulants) are useful fortreating or preventing naturally occurring immunosuppression orimmunosuppression from a variety of conditions and diseases. Compoundswhich depress immune responses (immunosuppressants) are useful fortreating or preventing, e.g., autoimmune diseases which arecharacterized by inflammatory phenomena and destruction of tissuescaused by the production, by the immune system, of the body's ownantibodies, or for suppressing rejection during, eg., tissue or organtransplantation. Compounds which inhibit cell proliferation are usefulfor treating conditions characterized by cell hyperproliferation, e.g.,as antineoplastic agents. Compounds of the invention are also useful asresearch tools, e.g., to investigate cell signaling.

In accordance with a preferred embodiment, the present inventionincludes methods of treating patients suffering from depressed immunesystems resulting from, e.g., chemotherapy treatment, radiationtreatment, radiation sickness, or HIV/AIDs; conditions associated withprimary B-cell deficiency (such as, e.g., Bruton's congenitala-γ-globulinemia or common variable immunodeficiency) or primary T-celldeficiency (such as, e.g., the DiGeorge and Nezelof syndromes, ataxiatelangiectasia or Wiskott-Aldrich syndrome); severe combinedimmunodeficiency (SCID), etc.; with an immunostimulant of the invention.The immunostimulants can also be used for vaccines (e.g.,anti-bacterial, anti-fungal, anti-viral or anti-protozoiasis),particularly for patients having immunocompromised states; or foranti-neoplastic vaccines.

In another preferred embodiment, the invention includes methods oftreating patients suffering from autoimmune disorders, such as, e.g.,rheumatoid arthritis, glomerulonephritis, Hashimoto's thyroiditis,multiple sclerosis, T cell leukemia, systemic lupus erythematosus,myasthenia gravis, autoimmune hemolytic anemia, autoimmunethrombocytopenic purpura, type 1 diabetes, Chrohn's disease, Grave'sdisease, celiac disease, or the like, with an immunosuppressant of theinvention. Immunosuppressants of the invention are also useful fortreating tissue or organ transplant rejection, e.g., hyper-acute orchronic graft-vs-host disease, allograft or xenograft rejection, etc.

As mentioned, the compounds of the invention also inhibithyperproliferation of cells, e.g., they can exhibit anti-neoplasticactivity. As a result, the inventive compounds are useful in thetreatment of a variety of conditions, e.g. cancers involving T cells andB cells. Among the types of cancer which can be treated with compoundsof the invention are e.g., leukemias, lymphomas, ovarian cancer andbreast cancer.

Compounds of the invention can be attached to an agent that, e.g.,targets certain tumors, such as an antibody which is specific for atumor-specific antigen. In this manner, compounds of the invention canbe transported to a target cell in which they then can act. Thecompounds can be further attached to a conventional cytotoxic agent(such as a toxin or radioactivity). When the inventive molecule binds toits target, e.g., p56^(lck), it not only will inhibit the enzymaticactivity, but will also destroy the target, and/or the cell in which thetarget resides, by means of the toxin.

The preferred aspects include pharmaceutical compositions comprising acompound of this invention and a pharmaceutically acceptable carrierand, optionally, another active agent as discussed below; a method ofinhibiting or stimulating a p56^(lck) kinase, e.g., as determined by aconventional assay or one described herein, either in vitro or in vivo(in an animal, e.g., in an animal model, or in a mammal or in a human);a method of modulating an immune response, e.g., enhancing or inhibitingan immune reaction; a method of treating a disease state, e.g., anautoimmune disease, a neoplasm, etc.; a method of treating a diseasestate modulated by p56^(lck) kinase activity, in a mammal, e.g., ahuman, including those disease conditions mentioned herein.

The present invention also relates to useful forms of the compounds asdisclosed herein, such as pharmaceutically acceptable salts and prodrugsof all the compounds of the present invention. Pharmaceuticallyacceptable salts include those obtained by reacting the main compound,functioning as a base, with an inorganic or organic acid to form a salt,for example, salts of hydrochloric acid, sulfuric acid, phosphoric acid,methane sulfuric acid, camphor sulfonic acid, oxalic acid, maleic acid,succinic acid and citric acid. Pharmaceutically acceptable salts alsoinclude those in which the main compound functions as an acid and isreacted with an appropriate base to form, e.g., sodium, potassium,calcium, magnesium, ammonium, and chlorine salts. Those skilled in theart will further recognize that acid addition salts of the claimedcompounds may be prepared by reaction of the compounds with theappropriate inorganic or organic acid via any of a number of knownmethods. Alternatively, alkali and alkaline earth metal salts areprepared by reacting the compounds of the invention with the appropriatebase via a variety of known methods.

The following are further examples of acid salts that can be obtained byreaction with inorganic or organic acids: acetates, adipates, alginates,citrates, aspartates, benzoates, benzenesulfonates, bisulfates,butyrates, camphorates, digluconates, cyclopentanepropionates,dodecylsulfates, ethanesulfonates, glucoheptanoates, glycerophosphates,hemisulfates, heptanoates, hexanoates, fumarates, hydrobromides,hydroiodides, 2-hydroxy-ethanesulfonates, lactates, maleates,methanesulfonates, nicotinates, 2-naphthalenesulfonates, oxalates,palmoates, pectinates, persulfates, 3-phenylpropiionates, picrates,pivalates, propionates, succinates, tartrates, thiocyannates, tosylates,mesylates and undecanoates.

Preferably, the salts formed are pharmaceutically acceptable foradministration to mammals. However, pharmaceutically unacceptable saltsof the compounds are suitable as intermediates, for example, forisolating the compound as a salt and then converting the salt back tothe free base compound by treatment with an alkaline reagent. The freebase can then, if desired, be converted to a pharmaceutically acceptableacid addition salt.

The compounds of the invention can be administered alone or as an activeingredient of a formulation. Thus, the present invention also includespharmaceutical compositions of compounds of formulas I, II or IIIcontaining, for example, one or more pharmaceutically acceptablecarriers.

Numerous standard references are available that describe procedures forpreparing various formulations suitable for administering the compoundsaccording to the invention. Examples of potential formulations andpreparations are contained, for example, in the Handbook ofPharmaceutical Excipients, American Pharmaceutical Association (currentedition); Pharmaceutical Dosage Forms: Tablets (Lieberman, Lachman andSchwartz, editors) current edition, published by Marcel Dekker, Inc., aswell as Remington's Pharmaceutical Sciences (Arthur Isol, editor),1553-1593 (current edition).

In view of their high degree of selective p56^(lck) kinase inhibition orstimulation, the compounds of the present invention can be administeredto anyone requiring p56^(lck) kinase inhibition or stimulation.Administration may be accomplished according to patient needs, forexample, orally, nasally, parenterally (subcutaneously, intravenously,intramuscularly, intrasternally, and by infusion) by inhalation,rectally, vaginally, topically and by ocular administration. Injectioncan be, e.g., intramuscular, intraperitoneal, intravenous, etc.

Various solid oral dosage forms can be used for administering compoundsof the invention including such solid forms as tablets, gelcaps,capsules, caplets, granules, lozenges and bulk powders. The compounds ofthe present invention can be administered alone or combined with variouspharmaceutically acceptable carriers, diluents (such as sucrose,mannitol, lactose, starches) and excipients known in the art, includingbut not limited to suspending agents, solubilizers, buffering agents,binders, disintegrants, preservatives, colorants, flavorants, lubricantsand the like. Time-release capsules, tablets and gels are alsoadvantageous in administering the compounds of the present invention.

Various liquid oral dosage forms can also be used for administeringcompounds of the inventions, including aqueous and non-aqueoussolutions, emulsions, suspensions, syrups, and elixirs. Such dosageforms can also contain suitable inert diluents known in the art such aswater and suitable excipients known in the art such as preservatives,wetting agents, sweeteners, flavorants, as well as agents foremulsifying and/or suspending the compounds of the invention. Thecompounds of the present invention may be injected, for example,intravenously, in the form of an isotonic sterile solution. Otherpreparations are also possible.

Suppositories for rectal administration of the compounds of the presentinvention can be prepared by mixing the compound with a suitableexcipient such as cocoa butter, salicylates and polyethylene glycols.Formulations for vaginal administration can be in the form of a pessary,tampon, cream, gel, paste, foam, or spray formula containing, inaddition to the active ingredient, such suitable carriers as are knownin the art.

For topical administration the pharmaceutical composition can be in theform of creams, ointments, liniments, lotions, emulsions, suspensions,gels, solutions, pastes, powders, sprays, and drops suitable foradministration to the skin, eye, ear or nose. Topical administration mayalso involve transdermal administration via means such as transdermalpatches.

Aerosol formulations suitable for administering via inhalation also canbe made. For example, for treatment of disorders of the respiratorytract, the compounds according to the invention can be administered byinhalation in the form of a powder (e.g., micronized) or in the form ofatomized solutions or suspensions. The aerosol formulation can be placedinto a pressurized acceptable propellant.

The compounds can be administered as the sole active agent or incombination with other pharmaceutical agents, such as other agents whichinhibit or stimulate tyrosine kinases, signal transduction processes,cell proliferation and/or immune responses. Inhibitory agents include,e.g., cyclosporine, FK506, rapamycin, leflunomide, butenamindes,corticosteroids, atomeric acid, dipeptide derivative, tyrphostin,Doxorubicin or the like. In such combinations, each active ingredientcan be administered either in accordance with its usual dosage range ora dose below its usual dosage range.

The dosages of the compounds of the present invention depend upon avariety of factors including the particular syndrome to be treated, theseverity of the symptoms, the age, sex and physical condition of thepatient, the route of administration, the frequency of the dosageinterval, the particular compound utilized, the efficacy, toxicologyprofile, pharmacokinetic profile of the compound, and the presence ofany deleterious side-effects, among other considerations.

By “effective dose” or “therapeutically effective dose” is meant herein,in reference to the treatment of a cancer, an amount sufficient to bringabout one or more of the following results: reduce the size of thecancer; inhibit the metastasis of the cancer; inhibit the growth of thecancer, preferably stop cancer growth; relieve discomfort due to thecancer; and prolong the life of a patient inflicted with the cancer.

A “therapeutically effective amount,” in reference to the treatment of ahyper-proliferative cell disorder other than a cancer refers to anamount sufficient to bring about one or more of the following results:inhibit the growth of cells causing the disorder, preferably stoppingthe cell growth; relieve discomfort due to the disorder; and prolong thelife of a patient suffering from the disorder.

A “therapeutically effective amount”, in reference to treatment of anautoimmune disorder refers to an amount sufficient to bring about one ormore of the following results: inhibit or ameliorate the symptoms of thedisease; inhibit progressive degeneration of cells involved in thedisorder; relieve discomfort due to the disorder; and prolong the lifeof a patient suffering from the disorder.

A “therapeutically effective amount”, in reference to treatment of apatient undergoing tissue or organ transplantation refers to an amountsufficient to bring about one or more of the following results: inhibitor prevent rejection of the transplanted material; relieve discomfortresulting from rejection of the transplait; and prolong the life of apatient receiving a transplant.

A “therapeutically effective amount,” in reference to treatment of animmunosuppressive patient refers to an amount sufficient to bring aboutone or more of the following results: increase the number of T cells ornumber of activated T cells; reduce the immuosuppressed state of thepatient; relieve discomfort due to the disorder; and prolong the life ofa patient suffering from the disorder.

The compounds of the invention are administered at dosage levels and ina manner customary for p56^(lck) kinase inhibitors or stimulators, orother analogous drugs, such as those mentioned above. For example,cyclosporine is administered (for transplants) at about 7.95±2.81mg/kg/day (see PDR(Physician's Desk Reference)); FK506 is administered(for transplants) at about 0.15-0.30 mg/kg/day (see PDR); and rapamycinis administered (for transplants) at about 2-6 mg/day, e.g., about 0.024mg/kg/day for an 81 kg adult (see Thomas A. Stargy TransplantationInstitute web site). See also, e.g., disclosures in U.S. Pat. Nos.5,688,824, 5,914,343, 5,217,999, 6,133,301 and publications citedtherein.

For example, compounds I and III of the invention can be administered,in single or multiple doses, at a dosage level of, for example, 1 μg/kgto 500 mg/kg of body weight of patient/day, preferably between about 100μg /kg/day and 25 mg/kg/day. For compound II, dosages are adjusted so asto generate an immunostimulatory or immunosuppressive effect, asdesired. A lower dosage (immunostimulatory) can be between about 1 μg/kg/day and 750 μg/kg/day, preferably between about 10 μg/kg/day and 500mg/kg/day. A higher dosage (immunosuppressive) can be between about 1mg/kg/day and 750 mg/kg/day, preferably between about 10 mg/kg/day and450 mg/kg/day.

In carrying out the procedures of the present invention it is of courseto be understood that reference to particular buffers, media, reagents,cells, culture conditions and the like are not intended to be limiting,but are to be read so as to include all related materials that one ofordinary skill in the art would recognize as being of interest or valuein the particular context in which that discussion is presented. Forexample, it is often possible to substitute one buffer system or culturemedium for another and still achieve similar, if not identical, results.Those of skill in the art will have sufficient knowledge of such systemsand methodologies so as to be able, without undue experimentation, tomake such substitutions as will optimally serve their purposes in usingthe methods and procedures disclosed herein.

In the foregoing and in the following examples, all temperatures are setforth uncorrected in degrees Celsius; and, unless otherwise indicated,all parts and percentages are by weight.

EXAMPLES Example 1 Jurkat Cell Activation-dependent Phosphorylation

Phosphorylation in Jurkat cells activated by the monoclonal antibody,OKT-3, is correlated with T-cell activation.

A. Compounds are tested for their effect on OKT-3 activated Jurkatcells. OKT-3 is a monoclonal antibody against CD3-ε chain. Treatment ofJurkat cells with OKT-3 antibody for 5 min at 37° C. activates Jurkatcells and induces tyrosine phosphorylation of several cellular proteins.In JCaM1.6 Jurkat cells expressing p56^(lck) with defective kinaseactivity, the OKT-3-mediated phosphorylation of cellular proteins doesnot take place, indicating that p56^(lck) plays an essential role inthis process. Experiments are carried out to determine if compounds ofthe invention, or known compounds which can serve as standards, affectcellular tyrosine phosphorylation stimulated by OKT-3 in Jurkat cells.For example, compounds II′, and III′ are shown to exhibit haveinhibitory activity at 100 μM, and compound I′ to exhibit stimulatoryactivity.

The assay is performed as follows: Jurkat cells (1×10⁶ cells) in RPMI1640 supplemented with 10% fetal bovine serum (FBS) are treated withOKT-3 antibody (0.2 μg) and compounds (100 μM) for 5 min at 37° C.Cultures are immediately placed on ice after the incubation period,washed 3 times with cold PBS and lysed in 20 μl of SDS-sample buffer.Samples are briefly sonicated and boiled for 5 min and applied toSDS-PAGE (12% gel). After SDS-PAGE, proteins are blotted onto ImmobilonP membrane (Millipore) for western blot analysis. Membranes are blockedovernight with 5% dried milk in TBST (Tris buffered saline containing 1%triton X-100). After washing, blots are incubated with monoclonalanti-phosphotyrosine antibody for 1 h in TBST followed by 1 hrincubation with horseradish peroxidase (HRP)-conjugated goat anti-mouseIgG. Blots are developed using enhanced chemiluminescence (ECL, Pierce).

B. Dose response experiments are performed. For example, compound I′ isshown to significantly stimulate tyrosine phosphorylation of Jurkatcellular proteins in a dose-dependent manner. Compound II′ stimulatestyrosine phosphorylation of cellular proteins at a lower dose (0.1 and 1μM), but inhibits phosphorylation at the higher dose (100 μM). CompoundIII′ completely inhibits tyrosine phosphorylation of cellular proteins.

The assay is performed as follows: Western blot analyses usinganti-phosphotyrosine antibody are performed. Compounds (e.g., inventivecompounds I′, II′ and III′) are added at concentrations of 0.1, 1, 10and 100 μM. Cells are cultured as above. Compound I′ is shown to inducedose-dependent stimulation of tyrosine phosphorylation, while CompoundIII′ inhibits tyrosine phosphorylation even at the lowest concentration(0.1 μM tested. Compound II′ gives a biphasic response, and stimulatestyrosine phosphorylation at lower doses (0.1 and 1 μM) while inhibitingphosphorylation at a higher dose (100 μM).

Example 2 Effect on Jurkat Cell Viability

Compounds can be tested to determine whether they are cytotoxic orcytostatic. To test whether a compound which inhibits OKT-3-mediatedstimulation of tyrosine phosphorylation has a cytotoxic effect on Jukatcells, its effect on the growth of Jurkat cells is tested. For example,compound III′ at 10 μM is shown to exhibit some cytostatic effect,inhibiting the growth of Jurkat cells, but no cytotoxic effect isobserved. Jurkat cells resume their normal rate of growth 24 hours afterthe addition of compound III′, indicating that the effect of compoundIII′ on growth is reversible.

Example 3 Inhibition of IL-2 Production in Jurkat Cells

Interleukin 2 (IL-2) is an autocrine growth factor for T cells, theproduction of which requires T cell antigen receptor and co-receptorgenerated activation signals in normal T cells. The production of IL-2is a hallmark of T cell activation signaling, leading to the clonalexpansion of antigen-specific T cell clones. In Jurkat cells, treatmentof cells with OKT-3 leads to the production of IL-2. In order to testwhether a compound which inhibits OKT-3 induced tyrosine phosphorylationof cellular proteins also inhibits IL-2 production, Jurkat cells aretreated with OKT-3 in the presence or absence of the compound. Forexample, about 50% inhibition of IL-2 production is observed when cellsare treated with 10 μM compound III′ at the time of OKT-3 treatment. Atlower concentrations, compound III′ has no observable effect. Unlike thecomplete inhibition of OKT-3 induced tyrosine phosphorylation, theinhibition of IL-2 production by compound III′ is partial.

The assay is performed as follows: Jurkat cells (1×10⁶ cells) aretreated with OKT-3 antibody (0.2 μg) in the presence or absence ofcompounds (e.g., Compound III′) (0.1, 1 and 10 μM) for 24 h in RPMI1640+10% FBS. At the end of the incubation period, culture media areharvested and assayed for human IL-2 by RIA. Cells treated with OKT-3only and PMA+Ionomycin serves as a positive control. Untreated cellsserve as a negative control. IL-2 production is not detected inuntreated cells. Cells treated with 10 μM of Compound III′ are shown toinhibit IL-2 production by 44%. The standard deviation is 0.23 pg/ml.

Example 4 Binding to the SH2 Domain Using [³H]-compound

Purified recombinant P56^(lck)-SH2 domain expressed as a GST-fusionprotein in bacteria is bound to either anti-GST agarose beads orglutathione-agarose beads. These beads bind the GST-p56^(lck)-SH2protein and facilitate the separation of [³H]-test compound bound SH2domain from unbound compound. Alternatively, dextran-coated activatedcharcoal solution is used to separate bound from unbound compounds.

-   -   Determination of K_(D): The binding affinity of a compound is        determined by applying standard Scatchard analysis where the        binding assay is performed in the presence of a fixed amount of        the [³H]-compound and an increasing amount of cold compound. The        K_(D) of the compound is calculated using the Ligand Program        (Munson & Rodbard 1980), Analytical Biochem., 107, 220-239. The        co-P is evident from, e.g., Sun et al., 1987, Biochem. Biophys.        Res. Comm. 148, 603-608.    -   Determination of IC₅₀: In addition to the determination of        K_(D), the IC₅₀ value for the compounds to inhibit p56^(lck)-SH2        domain binding to the N-terminal pY of CD3 ζITAM2 is measured.        The SH2 domain of p56^(lck) has the highest binding affinity        (0.1 μM) to the N-terminal pY of the second ITAM of CD3ζ chains.        A synthetic peptide corresponding to this region is made and        conjugated to agarose beads (ζ-NpY-ITAM2-agarose). P56^(lck)        binds to ζ-NpY-ITAM2-agarose through its SH2 domain and can be        precipitated. Using the recombinant p56^(lck) GST-SH2 protein,        binding assays using ζ-NpY-ITAM2-agarose are carried out in the        presence or absence of the compounds at various concentrations        (0.01˜100 μM) in RIPA buffer. After 2 h of the binding reaction        at room temperature, beads are harvested and washed three times        with RIPA buffer. The relative amounts of GST-SH2 bound to the        ζ-NpY-ITAM2-agarose are measured by a colorimetric assay after        incubating the beads with anti-GST antibody and HRP-conjugated        secondary antibody and HRP enzyme substrate. The OD read out        from the binding of GST-SH2 to the ζ-NpY-ITAM2-agarose in the        absence of the compound serves as positive control and        represents the 100% bound level. Background (ζ-NpY-ITAM2-agarose        without GST-SH2) is subtracted front each value. Assays are        carried out in triplicate and the average from three independent        assays are used to calculate IC₅₀ values for each compound.

Example 5 Co-immunoprecipitation Experiments

The binding of compounds to the p56^(lck) SH domain pY+3 pocket affectsthe SH2-mediated interaction of p56^(lck) with phosphotyrosine residuesof cellular target proteins. For example, the treatment of cells with aninhibitor compound inhibits the association of p56^(lck) with CD3ξ andZAP-70, whereas an increase in the association occurs followingtreatment with a stimulator compound. The presence of these molecularinteractions is assessed by co-immunoprecipitation assays in activatedJurkat cells in the presence or absence of the compounds. Using thisassay (in conjunction with p56^(lck) kinase assays as describedelsewhere herein), one can estimate the IC₅₀ of a compound for blockingthe molecular interactions and the ED₅₀ of a compound for activatingkinase activity. These values are generally in the vicinity of the K_(D)of the compound binding to the SH2 domain, provided that the effect ofthe compound is mediated by binding to the SH2 domain of p56^(lck).

Jurkat cells (5×10⁶ to 1×10⁷/ml; 1 ml/condition) are activated usingOKT-3 antibody (1 μg/5×10⁶ cells) in the presence or absence of thecompound to be tested (0.01 to 100 μm). Following 10 min of activationat 37° C., cells are harvested and lysed using RIPA or NP-40 lysisbuffer. After removing insoluble materials, the supernatant is treatedwith 1 to 2 μg of antibody against either CD3ξ or ZAP-70. Immune complexis precipitated using Protein A or G conjugated agarose beads (10 μl of50% slur) at 4° C. Beads are harvested and washed three times with lysisbuffer, boiled for 2 min., resolved on SDS PAGE (e.g., 12.5%) andblotted on Immobilon-P membrane for Western blot analysis, usinganti-p56^(lck) antibody. Samples from non-activated cells and activatedcells in the presence of the compound serve as negative and positivecontrols. Blots are also re-probed with the precipitating antibody toensure that equal amounts of protein (either CD3ξ or ZAP-70)precipitates from each sample. The presence of co-immunoprecipitatingp56^(lck) is examined. A semi-quantitative analysis can also be carriedout by altering the compound concentrations in the assay and measuringthe relative amount of co-immunoprecipitating p56^(lck). Quantitation isperformed by performing EIA (using spectrophotometric assay followingHRP-conjugated 2° antibody+chromogenic substrate), chemoluminescence(using image analyzer following HRP-conjugated 2° antibody+ECL) orphosphorimage analysis (using [¹²⁵I]-2° antibody). IC₅₀ values for eachcompound are determined.

Example 6 Demonstration of Specificity of Binding

Based on sequence homology and other considerations five additional SH2domain containing kinases, including ZAP-70, which contains two pYbinding sites, are selected for testing of specificity, bothcomputationally and experimentally. These proteins are listed in Table1:

TABLE 1 SH2 domains used for determination of selectivity SH2 domain EFloop FB loop βG strand PDB Entry lck Ser-Pro-Arg Pro Arg-Pro-Cys 1LKKHck Ser-Pro-Arg Ser Val-Pro-Cys 1QCF Fyn Thr-Thr-Arg Glu Val-Pro-Cys1AOT Src Thr-Ser-Arg Ser Asn-Val-Cys 1BKL Shc Lys-Asp Glu Gln-Pro-Val1TCE ZAP-70, N^(a) Ala-Gly-Gly Cys Lys-Pro-Cys NA^(b) ZAP-70, C^(a)Pro-Glu-Gly Asp Glu-Ala-Cys NA^(b) ^(a)ZAP-70 contains 2 phosphotyrosinebinding sites in the N and C-terminal regions, designated ZAP-70, N andZAP-70, C, respectively ^(b)NA: Not available from the PDB, however, thestructure was obtained by the authors.

Specificity is determined via differential binding of the SH2 domains inTable 1 with the phosphopeptides listed in Table 2. The SH2 domains areselected based on homology with P56^(lck) as well as the availability of3D structal data. Phosphopeptides are selected based on their SH2 domainspecificity. The ζ-ITAM-2-C and ζ-ITAM-1 peptides are specific forp56^(lck) and ZAP-70, respectively. All Src kinases including p56^(lck)and Shc are known to bind with similar affinity to the hamster polyomamiddle T antigen peptide that contains the pYEEI sequence.

TABLE 2 Phosphopeptides used in the selectivity binding experiments pYPeptide SH2 domain SEQ ID No. 1 TATEGQpYQPQP (ζ-ITAM-2-C) p56^(lck) SEQID No. 2 GQNQLpYNELNLGRREEpYDVLDKR (ζ-ITAM-1) ZAP-70 SEQ ID No. 3EQpYEEIPIA (hamster polyoma middle T) p56^(lck), Src, Hck, Shc, Fyn

A solid phase binding competition assay between the compounds and thephosphopeptides is used. SH2 domains are PCR amplified and expressed asGST-fusion proteins in E. coli. The GST-fusion protein is purfied on aglutathione column. SH2 domains are cleaved off using thrombin andpurified on a gel filtration column. Synthetic phosphotyrosinecontaining peptides listed in Table 2 are synthesized using conventionalmethods. These peptides are biotinylated at the N-terminus, away fromthe SH2 docking site, using kits available from Pierce (Rockford, Ill.).A 96-well EIA plate is coated with a purified SH2 domain (1 μg/well;˜100 nmol). Biotinylated peptide (˜200 nmol) in the presence or absenceof varying concentrations (pmol to μmol) of compound is added.Biotinylated peptide bound to the SH2 domain is then measured in variousways, such as by colorimetric assay using HRP-streptavidin+substrate, byfluorescence using FITC-labeled streptavidin or by scintillation counterusing [¹²⁵I]-labeled streptavidin. OD (or cpm) reading of ζ-ITAM2-Cpeptide without compound constitutes 100% bound. OD (or cpm) reading ofSH2 only constitutes the blank. IC₅₀ values for each compound aredetermined. Alternatively, the assay is adapted to useglutathione-agarose beads to separate SH2 domain after the incubationperiod. K_(D) for each compound is determined using binding competitionassay.

Example 7 Mixed Lymphocyte Culture Assay

Another readout assay that is used to measure biological response is amixed lymphocyte culture assay in which lymphocytes from two differentstrains of mice with different histocompatibility antigens are mixed.Due to the difference in the histocompatibility antigens, resting Tcells from both strains of mice undergo blast transformation andpropagate. As in any T cell activation process, the activation ofp56^(lck) is essential. Therefore, the modulation of p56^(lck) activitycan be quantified as the downstream modulation in the levels of [³H]-TDRincorporation into DNA.

Lymph node or splenic lymphocytes are harvested from two differentallogeneic strains of mice. Cells (1×10⁶) from each strain are mixed in96 well cultureplates containing 200 μl of culture medium in thepresence or absence of the compound (0.1˜100 μM) and cultured for 72 h.Six hours before harvest, 0.5 μCi of [³H]-TdR is added to each well. Atthe end of the culture period, cells are harvested on a glass fiberfilter using a cell harvester. Filters are washed with PBS and then withethanol and [³H]-TdR incorporated into DNA is measured usingscintillation counting. Experiments are carried out in triplicate. Cellscultured in the absence of the compound serve as a positive control.Cells from each strain of mouse cultured in the absence of allogeneiclymphocytes serve as the negative control. The compounds are added every12 h. The compounds that inhibit protein phosphorylation and IL-2production have a similar effect on [³H]-TdR incorporation.

Example 8 Inhibition in vivo of Immune Response of Delayed-TypeHypersensitivity (DTH) and Anti-type II Collagen-induced RheumatoidArthritis in Mice

One experimental model is DTH response to PPD. DTH reaction is a typicalT cell immune response and, thus well suited to be used for assessingthe in vivo effect of the compounds. Mice are immunized using BCG incomplete Freund's adjuvant (CFA). After the initial immunization, atuberculin skin test is performed. Bilateral regions of BCG immunizedmouse skin are treated with hair removal cream. Interdermal injection oftuberculin is administered in two hair-removed sites. The compounddissolved in DMSO is applied to one of the sites every 12 h. The othersite is treated with DMSO only and serves as an internal positivecontrol. After one week, diameters of DTH reactions for both control andtreated sites are measured. Five mice are used in a group. Statisticalanalysis is carried out to determine effective dose required for 50%reduction in the DTH skin test over control. Treatment is carried outwith animals anesthetized (using vapolizer, 2% isoflurane). Animals arekept under anesthesia till DMSO is completely absorbed to the skin. Theamount of DMSO applied to each site is kept minimal (˜10 μl).

In addition to the DTH reaction, experiments are performed usingcollagen-induced arthritis (CIA) in mice. CIA is an experimental modelfor rheumatoid arthritis (RA) in human. A commercial kit is available toreliably induce CIA in mice in a short period of time. Monoclonalantibody cocktail against type II collagen is injected into mice, i.v.RA develops within 24-48 h after injection of the antibody cocktail andexacerbated swelling of the two hind paws becomes evident by day 6. Oneof the paws from each aal is treated with the compound dissolved inDMSO. The other paw is treated with DMSO only and serves as an internalpositive control. Swelling of the paws is measured. Thickness of thepaws from saline injected, DMSO-treated control mice serves as negativecontrol. Hind paws are dipped in DMSO solution (with or without thecompound to be tested) for 10 seconds twice daily at 12 h intervals.Animals are kept under anesthesia till DMSO is completely absorbed. Fivemice are used in a group. Statistical analysis is carried out todetermine the effective dose required for 50% reduction in the swellingof paws over the control for the inhibitory compounds. Treatment iscarried out using mice under anesthesia.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make changes andmodifications of the invention to adapt it to various usage andconditions.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The preceding preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

The entire disclosure of all applications, patents and publications,cited above and in the figures are hereby incorporated in their entiretyby reference.

1. A method of modulating the binding of a p56^(lck) molecule via an SH2 domain thereof to a cellular binding protein selected from the group consisting of CD3 chains, ZAP-70, p62, Lad, CD45, and Sam68, or modulating the activity of a p56^(lck) molecule via an SH2 domain thereof, wherein said method achieves the treatment of a disorder treated by immunostimulation and comprises administering to a patient in need thereof an effective amount of a compound of formula

or a pharmaceutically acceptable salt thereof, and binding to an SH2 domain of said p56^(lck) molecule by the compound of formula I′ or the pharmaceutically acceptable salt thereof.
 2. A method of modulating the binding of a p56^(lck) molecule via an SH2 domain thereof to a cellular binding protein selected from the group consisting of CD3 chains, ZAP-70, p62, Lad, CD45, and Sam68, or modulating the activity of a p56^(lck) molecule via an SH2 domain thereof, wherein said method achieves the treatment of a disorder treated by immunosuppression, and comprises administering to a patient in need thereof an effective amount of a compound of formula

or a pharmaceutically acceptable salt thereof, and binding to an SH2 domain of said p56^(lck) molecule by the compound of formula II′ or the pharmaceutically acceptable salt thereof.
 3. A method of claim 2, which achieves the treatment of a hyperproliferative cell disorder other than cancer by administering the compound of formula II′ in an effective amount to a patient in need thereof.
 4. A method of claim 2, which achieves the treatment of an autoimmune disorder by administering the compound of formula II′ in an effective amount to a patient in need thereof.
 5. A method of claim 2, which achieves the treatment of a tissue or organ rejection in a patient undergoing tissue or organ transplantation by administering the compound of formula II′ in an effective amount to a patient in need thereof.
 6. A method according to claim 2, which achieves the treatment of rheumatoid arthritis, glomerulonephritis, Hashimoto's thyroiditis, systemic lupus erythematosus, multiple sclerosis, T cell leukemia, myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, type 1 diabetes, Chrohns disease, Graves disease, celiac disease, hyper acute or chronic graft-vs-host disease, or an allograft or xenograft rejection by administering in an effective amount the compound of formula II′ to a patient in need thereof of treatment of rheumatoid arthritis, glomerulonephritis, Hashimoto's thyroiditis, systemic lupus erythematosus, multiple sclerosis, T cell leukemia, myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, type 1 diabetes, Chrohns disease, Graves disease, celiac disease, hyper acute or chronic graft-vs-host disease, or an allograft or xenograft rejection.
 7. A method of modulating the binding of a p56^(lck) molecule via an SH2 domain thereof to a cellular binding protein selected from the group consisting of CD3 chains, ZAP-70, p62, Lad, CD45, and Sam68, or modulating the activity of a p56^(lck) molecule via an SH2 domain thereof, wherein said method achieves the treatment of a disorder treated by immunosuppression, and comprises administering to a patient in need thereof an effective amount of a compound of formula

or a pharmaceutically acceptable salt thereof, and binding to an SH2 domain of said p56^(lck) molecule by the compound of formula III′ or the pharmaceutically acceptable salt thereof.
 8. A method of claim 7, which achieves the treatment of a hyperproliferative cell disorder other than cancer by administering the compound of formula III′ in an effective amount to a patient in need of treatment of a hyperproliferative cell disorder other than cancer.
 9. A method of claim 7, which achieves the treatment of an autoimmune disorder by administering the compound of formula III′ in an effective amount to a patient in need of treatment of an autoimmune disorder.
 10. A method of claim 7, which achieves the treatment of a tissue or organ rejection in the patient undergoing tissue or organ transplantation by administering a compound of formula III′ in an effective amount to a patient in need of treatment of a tissue or organ rejection.
 11. A method according to claim 7, which achieves the treatment of rheumatoid arthritis by administering the compound of formula III′ in an effective amount to a patient in need of treatment of rheumatoid arthritis.
 12. A method according to claim 7, which achieves the treatment of rheumatoid arthritis, glomerulonephritis, Hashimoto's thyroiditis, systemic lupus erythematosus, multiple sclerosis, T cell leukemia, myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, type 1 diabetes, Chrohns disease, Graves disease, celiac disease, hyper acute or chronic graft-vs-host disease, or an allograft or xenograft rejection by administering in an effective amount the compound of formula III′ to a patient in need thereof of treatment of rheumatoid arthritis, glomerulonephritis, Hashimoto's thyroiditis, systemic lupus erythematosus, multiple sclerosis, T cell leukemia, myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, type 1 diabetes, Chrohns disease, Graves disease, celiac disease, hyper acute or chronic graft-vs-host disease, or an allograft or xenograft rejection.
 13. A method of treating rheumatoid arthritis, glomerulonephritis, Hashimoto's thyroiditis, systemic lupus erythematosus, multiple sclerosis, T cell leukemia, myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, type 1 diabetes, Chrohns disease, Graves disease, celiac disease, hyper acute or chronic graft-vs-host disease, or an allograft or xenograft rejection, comprising administering to a patient in need thereof the compound of formula

or a pharmaceutically acceptable salt thereof.
 14. A method according to claim 13, which achieves the treatment of glomerulonephritis, Hashimoto's thyroiditis, or systemic lupus erythematosus by administering the compound of formula II′ or III′ in an effective amount to a patient in need of treatment of glomerulonephritis, Hashimoto's thyroiditis, or systemic lupus erythematosus.
 15. A method according to claim 13, which achieves the treatment of multiple sclerosis by administering the compound of formula II′ or III′ in an effective amount to a patient in need of treatment of multiple sclerosis.
 16. A method according to claim 13, which achieves the treatment of T cell leukemia by administering the compound of formula II′ or III′ in an effective amount to a patient in need of treatment of T cell leukemia.
 17. A method according to claim 13, which achieves the treatment of myasthenia gravis by administering the compound of formula II′ or III′ in an effective amount to a patient in need of treatment of myasthenia gravis.
 18. A method according to claim 13, which achieves the treatment of autoimmune hemolytic anemia, or autoimmune thrombocytopenic purpura by administering the compound of formula II′ or III′ in an effective amount to a patient in need of treatment of autoimmune hemolytic anemia, or autoimmune thrombocytopenic purpura.
 19. A method according to claim 13, which achieves the treatment of type 1 diabetes by administering the compound of formula II′ or III′ in an effective amount to a patient in need of treatment of type 1 diabetes.
 20. A method according to claim 13, which achieves the treatment of Chrohns disease, or Graves disease by administering the compound of formula II′ or III′ in an effective amount to a patient in need of treatment of Chrohns disease, or Graves disease.
 21. A method according to claim 13, which achieves the treatment of celiac disease by administering the compound of formula II′ or III′ in an effective amount to a patient in need of treatment of celiac disease.
 22. A method according to claim 13, which achieves the treatment of hyper acute or chronic graft-vs-host disease, or an allograft or xenograft rejection by administering the compound of formula II′ or III′ in an effective amount to a patient in need of treatment of hyper acute or chronic graft-vs-host disease, or an allograft or xenograft rejection.
 23. A method of treating an immunosuppressive disorder, comprising administering to a patient in need thereof the compound of formula

or a pharmaceutically acceptable salt thereof. 